Method and apparatus for measuring cell counts of Methanogens or methane producing activity thereof

ABSTRACT

A method for measuring cell counts or methane producing activity of Methanogens in an object for examination containing therein Methanogens, in which excited light of a particular wavelength region is irradiated onto the object for examination, and intensity of fluorescence of a particular wavelength region, which the object for examination radiates, is measured. Since, according to this invention, intensity of fluorescence specific to Methanogens is measured, it is possible to measure with high precision the cell counts or the methane producing activity of Methanogens existing in a multitude of microorganism groups and foreign substances such as digested sludge, etc. in a methane fermentation tank, etc. of a sewage treatment system, in particular.

FIELD OF TECHNOLOGY

This invention relates to a method and apparatus for measuring cellcounts of Methanogens or methane producing activity thereof in an objectfor examination having Methanogens therein. More particularly, it isconcerned with a method which is applicable to measurement of the cellcounts of Methanogens or the methane producing activity of suchmicroorganism existing in a multitude of microorganism groups and inforeign substances such as digested sludge, and so forth which are heldin a methane fermentation tank of a sewage treatment system, or thelike.

BACKGROUND OF TECHNOLOGY

FIG. 1 shows a conventional apparatus for conducting this type ofmeasurment. In the drawing, a reference numeral 1 designates an objectfor examination; a numeral 2 refers to a light source; 3 denotes a powersource for applying an electric potential to the light source 2; 4represents a photoelectric multiplying tube; 5 a power source forapplying an electric potential to the photoelectric multiplying tube 4;and 6 indicates a detector for measuring photo-current of thephotoelectric multiplying tube 4.

Light emitted from the light source 2 passes through the object forexamination 1 containing therein microorganism. The transmitted light isreceived by the photoelectric multiplying tube 4, and its intensity ismeasured by the detector 6 as a photocurrent value of the photoelectricmultiplying tube 4. Since there is established a definite relationshipbetween absorbance and concentration of microorganism existing in theabovementioned object for examination 1, thus obtained, when a visiblelight is used as the light source, the concentration of microorganismcan be evaluated by measuring the absorbance of the object forexamination. As the result of, or, in connection with, this, the cellcounts can be evaluated.

Also, as an other method for measuring the microorganism activity, therehas been known a method for optical measurement of a quantity ofbiological substance relative to the energy metabolism which is called"ATP" (Adenosine Triphosphate) or "NAD(P)H" (Nicotinamide AdenineDinucleotide(phosphate)) contained in microorganism.

As mentioned in the preceding, the conventional method for measuring thecell counts or the activity of microorganism is to measure theabsorbance of an object for examination 1, on account of which suchmethod is effective only if the object for examination 1 is composed ofone kind of microorganism and contains no foreign substance such assludge, etc. However, if the object for examination 1 is composed ofmany kinds of microorganisms and, moreover, if foreign substances arecontained within the examined object, it was impossible to selectivelymeasure the cell counts or activity of a particular kind ofmicroorganism. Further, since ATP and NAD(P)H are biological substancesexisting in all kinds of microorganisms, the method is not suitable formeasuring the cell counts or methane producing activity of Methanogensalone.

SUMMARY OF THE INVENTION

The present invention is an apparatus and method for measuring the cellcounts or the methane producing activity of the above-mentionedMethanogens, by irradiating excited light of a particular wavelengthrange onto an object for examination containing therein Methanogens, andmeasuring the intensity of fluorescence of a particular wavelengthregion which the above-mentioned object for examination radiates. Moreparticularly, the invention aims at providing a method and apparatuscapable of measuring the cell counts or the methane producing activityof the above-mentioned Methanogens even if there is a mixed system ofmicroorganism containing therein foreign substances such as digestedsludge, etc. as in a methane fermentation tank.

According to the present invention, it is possible to determine withhigh precision the cell counts or the methane producing activity ofMethanogens by use of light in a wavelength range of from 220 nm to 310nm, or from 220 nm to 255 nm, or from 260 nm to 305 nm, or from 380 nmto 440 nm, as the excited light of a particular wavelength range to beirradiated onto the object for examination which contains thereinMethanogens.

According to the present invention, it is possible to accurately measurethe cell counts or the methane producing activity of Methanogens bymeasuring the intensity of the fluorescence in a wavelength range offrom 330 nm to 370 nm, or from 450 nm to 490 nm, which the examiningobject radiates.

According to the present invention, it is possible to increase themeasuring sensitivity by rendering the object for examination to bealkaline, or substituting solution which does not emit fluorescence of arange of the measuring wavelength in the excited wavelength range forthe liquid component of the examining object, or diluting the object forexamination.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a block diagram for explanation of the conventional method formeasuring cell counts of microorganism;

FIG. 2 is a block diagram for explanation of a method for measuring thecell counts or the methane producing activity of Methanogens accordingto one embodiment of the present invention;

FIG. 3 is a graphical representation showing the fluorescence propertiesof a component model having components other than Methanogens in thedigested sludge;

FIGS. 4 and 8 are graphical representations showing the fluorescenceproperties of Methanogens;

FIGS. 5 and 9 are graphical representations showing the fluorescenceproperties of digested sludge containing Methanogens;

FIGS. 6 and 7 as well as FIGS. 11 and 12 are respectively graphicalrepresentations showing interrelationship between the cell counts ofMethanogens and the intensity of excited spectrum of fluorescence aswell as between the methane production quantity and the intensity ofexcited spectrum of fluorescence;

and FIG. 10 is a characteristic curve showing variations in thefluorescence properties of Methanogens and Escherichia coli due to pHvalue.

PREFERRED EMBODIMENT OF THE INVENTION

In the following, the present invention will be described with referenceto one embodiment as shown in the drawing. Referring now to FIG. 2, areference numeral 8 designates the interior of a methane fermentationtank containing an object for examination; a numeral 9 refers to opticalfibers to introduce and take out light into and from the fermentationtank 8; a numeral 10 denotes a light collecting device for collectinglight emitted from a light source 13 into one of the optical fibers 9; anumeral 11 represents a selector to adjust intensity of light in thelight source 13; a numeral 12 referes to an optical filter to limit awavelength of light emitted from the light source 13; a numeral 14indicates a power source for the light source; a numeral 15 represents alight collecting device for collecting light emitted from the otheroptical fiber 9; a numeral 16 refers to an optical filter to limit awavelength of light at the light receiving side; a numeral 17 designatesa photoelectric multiplying tube; a numeral 18 denotes a power sourcefor the photoelectric multiplying tube; and a numeral 19 represents adetector for measuring photo-current in the photoelectric multiplyingtube 17.

In the following, explanations will be given as to the principle and thefunction of the present invention. It has been known that Methanogenshas physiological properties different from that of ordinarymicroorganisms and specific which are to it, although its electrontransport system which takes part in the energy metabolism ofMethanogens has not yet been clarified in its entirety. It is knownthat, in this electron transport system existing in the energymetabolism of the Methanogens, a substance called "Factor₄₂₀ (F₄₂₀)"functions as the electron carrier, which is a substance found inMethanogens and which is not existent in other biological bodies.Therefore, if the substance, which contain therein this substance F₄₂₀as the principal component and takes part in the electron transportsystem of Methanogens possesses its physicochemical properties which arepeculiar to it and and measurable, and are different from those of themicroorganism groups other than Methanogens and foreign substances inthe object for examination such as digested sludge, etc., and, moreover,if such properties are measurable in the state of viable cell (i.e.,microorganism in its living state), such substance can be used as aparameter in measuring the cell counts or the methane producing activityof Methanogens. In particular, since the substance F₄₂₀, which takespart in the electron transport system of Methanogens as the principalcomponent, it is directly related to the methane producing mechanism inits physiological function. Hence it can be an effective object, in themeasurement of the methane producing activity.

As the result of strenuous efforts in studying and research done by thepresent inventor on the basis of this phenomenon, it has been clarifiedthat the fluorscent property which is considered to be due to thesubstance F₄₂₀ in Methanogens takes different behaviour in the state ofcell from the fluorescence properties to be ascribable to microorganismsother than Methanogens and to foreign substances in the digested sludge.Based on this clarification, the present invention has been completed.

FIG. 3 indicates fluorescence excitation spectrum and fluorescencespectrum of Escherichia coli suspended in a nutrient medium (composed of10 g/l of trypton, 10 g/l of sodium chloride, and 5 g/l of yeastextract). In the graphical representation, the fluorescence excitationspectrum indicates intensity of fluorescence in the wavelength of 470 nmwith respect to changes in the excited wavelength, while thefluorescence spectrum indicates such fluorescence spectrum in theexcited wavelength of 380 nm. The substances which radiate fluorescencein various biological substances, amino acids such as tryptophan,tyrosine, phenylalanine, and so forth are representative. However, thosespecimens used in this invention as the object for examination are allmixtures of these fluorescent substance, hence they may be regarded as amodel of a biological specimen series other than Methanogens.

FIG. 4 shows fluorescence excitation spectrum and fluorescence spectrumof Methanogens (here, it is Methanosarcina barkeri) suspended in aminimum medium (a culture medium not containing therein any biologicalcarbon source). In this case, since the minimum medium is used, nofluorescence from the culture medium can be observed. Therefore, thefluorescence properties shown in FIG. 4 is derived from Methanogensalone. As is apparent from comparison between FIGS. 3 and 4, thefluorescence properties of Methanogens takes different behaviour fromthe fluorescence properties of the specimen as used for the purpose ofFIG. 3, i.e., the model specimen of the microorganism other thanMethanogens and foreign substances.

FIG. 5 indicates fluorescence excitation spectrum and fluorescencespectrum of digested sludge sampled from a methane fermentation tank.Upon comparison of FIGS. 4 and 5, it will be seen that a well-matchedbehaviour is exhibited with the excitation light having a wavelengthrange of from 220 nm to 310 nm (in particular, having the respectivepeaks in the wavelength ranges of from 220 nm to 255 nm and from 260 nmto 305 nm) and the fluorescence having its wavelength range of from 330nm to 370 nm, so that the fluorescence properties of the digested sludgein the above-mentioned wavelength range is ascribable to Methanogens.Also, it cannot be decided that, of the fluorescence properti,es shownin FIG. 5, the fluorescence spectrum of a wavelength range of from 380nm to 450 nm is ascribable to Methanogens, because it is overlappingwith the behaviour of the model of the biological specimen series otherthan Methanogens as shown in FIG. 3. Further, the fluorescence spectrumhaving its peak at the wavelength of 500 nm shown in FIG. 5 appears tobe the fluorescence light from the component other than Methanogens inthe digested sludge as the result of comparison between FIGS. 3 and 4.

FIG. 8 indicates the fluorescence excitation spectrum and thefluorescence spectrum of Methanogens (here, it is Methanosarcinabarkeri) suspended in a minimum culture medium (a culture medium notcontaining therein organic carbon source). For the sake of comparison,fluorescence excitation spectrum and the fluorescence spectrum ofEscherichia coli suspended in the minimum medium are also shown. Itshould be noted here that the fluorescence excitation spectrum ofMethanogens indicates an intensity of fluorescence having a wavelengthof 470 nm with respect to changes in the excited wavelength, while thefluorescence spectrum indicates such fluorescence spectrum at theexcited wavelength of 400 nm. Further, the fluorescence excitationspectrum of Escherichia coli indicates an intensity of fluorescencehaving a wavelength of 470 nm with respect to changes in the excitedwavelength, while the fluorescence spectrum indicates such fluorescencespectrum at the excited wavelength of 400 nm. In this case, since theminimum medium is used, the fluorescence properties shown in FIG. 8 isderived from the microorganism alone, hence it is seen that thefluorescence properties of Methanogens takes a behaviour which isremarkably different from the fluorescence properties of Escherichiacoli. Furthermore, from comparison with FIG. 3, it can be understoodthat the fluorescence properties of Methanogens takes a differentbehaviour from the fluorescence properties of the model specimen ofmicroorganism other than Methanogens and foreign substances.

FIG. 9 shows the fluorescence excitation spectrum and the fluorescencespectrum of digested sludge sampled from a methane fermentation tank. Inthis case, the fluorescence excitation spectrum indicates an intensityof fluorescence having a wavelength of 470 nm with respect to changes inthe excited wavelength, while the fluorescence spectrum indicates suchfluorescence spectrum at the excited wavelength of 420 nm. On comparingFIGS. 8 and 9, it will be seen that a well-matched behaviour isexhibited at the fluorescence excitation spectrum in a wavelength rangeof from 380 nm to 440 nm, and at the fluorescence spectrum in awavelength range of from 450 nm to 490 nm, hence the fluorescenceproperties of the digested sludge in the above-mentioned wavelengthranges is derived from the Methanogens.

FIG. 10 shows the fluorescence excitation spectrum of Methanogens andEscherichia coli in the nutrient medium at the respective pH values. Thefluorescence excitation spectrum indicates an intensity of fluorescencehaving a wavelength of 470 nm with respect to changes in the excitedwavelength. From the graphical representation, it will be seen thatsubstantially no changes have taken place in the fluorescence excitationspectrum with Escherichia coli in the pH values ranging from 7 to 11,while the fluorescence excitation peak wavelength and its intensity varydepending on the pH value with Methanogens, the peak wavelength shiftingby about 20 nm to the long wavelength side at the pH value of 11 ascontrasted to the pH value 7, and the peak intensity also increasing ashigh as about three times. In this way, by addition of basic solution orbasic solid such as, for instance, sodium hydroxide, potassiumhydroxide, ammonium hydroxide, and so on, it becomes possible to renderthe object for examination to be alkaline in a pH value range of from 7to 14, to increase the signal intensity of a fluorescence signal derivedfrom Methanogens, to shift the excited fluorescence wavelength range tothe long wavelength side in a range of from zero to 30 nm, and toincrease the S/N ratio with respect to a background fluorescence derivedfrom those components other than Methanogens. In particular, as isapparent from FIGS. 8 to 10, when use is made of light having awavelength range of from 410 nm to 430 nm as the excited light and lighthaving a wavelength range from 460 nm to 480 nm as the fluorescence, thespectra of both fluorescence excitation and fluorescence can be measuredin the vicinity of their peak points.

It is further possible to increase the S/N ratio of the fluorescencesignal derived from Methanogens even by separating liquid component outof the solid and by substituting liquid components in the object forexamination with a solution such as water, etc. which does not radiatefluorescence of a measuring wavelength range in the excited wavelengthrange by use of the centrifugal operation or the filtration operation,or others; or diluting the object for examination with a solution suchas water, etc. which does not radiate fluorescence of a measuringwavelength range in the excited wavelength range. Further, in this case,if use is made of a basic solution such as, for example, sodiumhydroxide, potassium hydroxide, and so forth as the solution which doesnot radiate fluorescence of a measuring wavelength range in the excitedwavelength range, the effect of the above-mentioned alkalinity is alsoadded.

From the above-mentioned result of studies and researches, it has beenfound out that the following methods may be adopted for measuring thecell counts or methane producing activity of Methanogens existing in amultitude of microorganism groups and foreign substances such asdigested sludge, etc. in the methane fermentation tank, and so on.

(1) As the light for fluorescence excitation, use is made of lighthaving a wavelength range of from 220 nm to 310 nm, and the cell countsand the methane producing activity of Methanogens is determined from theinterrelationship between the intensity of the excitation spectrum andthe cell counts or the methane producing activity. Since the excitationspectrum of Methanogens has its peak values in the above-mentionedwavelength range, in particular, in the wavelength ranges of from 220 nmto 255 nm and from 260 nm to 305 nm, the cell counts or the methaneproducing activity of Methanogens can be determined with high precisionby measurement of the intensity of the excitation spectra in these twowavelength ranges.

(2) As the fluorescence, use is made of light having a wavelength rangeof from 330 nm to 370 nm, and the cell counts or the methane producingactivity of Methanogens is determined on the basis of theinterrelationship between the intensity of the fluorescence spectrum andthe cell counts or the methane producing activity of Methanogens.

(3) As the light for fluorescence excitation, use is made of lighthaving a wavelength range of from 380 nm to 440 nm, and the cell countsor the methane producing activity of Methanogens is determined on thebasis of the interrelationship between intensity of the excitationspectrum and the cell counts or the methane producing activity ofMethanogens.

(4) As the fluorescence, use is made of light having a wavelength rangeof from 450 nm to 490 nm, and the cell counts or the methane producingactivity of Methanogens is determined on the basis of theinterrelationship between intensity of the fluorescence spectrum and thecell counts or the methane producing activity of Methanogens.

(5) As the methods for increasing the S/N ratio of the fluorescencederived from Methanogens with respect to the background fluorescencederived form the components other than Methanogens, the following threemethods can be employed:

(a) the object for examination is rendered to be alkaline;

(b) the liquid component of the object for examination is substituted byother solution; and

(c) the object for examination is diluted.

The interrelationship between the intensity of the fluorescenceexcitation spectrum or the fluorescence spectrum and the cell counts orthe methane producing activity can be found from Methnomonas isolatedfrom digested sludge, such as Methnosarcina barkeri, as a referencespecimen. As examples of this, FIGS. 6 and 7 as well as FIGS. 11 and 12respectively indicate the interrelationship between the cell counts andthe intensity of the excitation spectrum as well as theinterrelationship between the methane producing quantity and theintensity of the excitation spectrum. By the way, FIGS. 6 and 7 indicatethe characteristics when use is made of light having a wavelength of 280nm as the light used for the excitation, and light having a wavelengthof 350 nm occurs as the fluorescence, while FIGS. 11 and 12 indicate thecharacteristics when use is made of light having a wavelength of 420 nmas the light used for the excitation, and the light having a wavelengthof 470 nm occurs as the fluorescence.

According to such method of measurement, the cell counts or the methaneproducing activity of Methanogens can be measured real time duringoperation of the methane fermentation tank, hence remarkable effect canbe expected in controlling operations of the methane fermentation tank.

Further, much convenience can be secured when the measuring system isconstructed as shown in FIG. 2. That is to say, when a system controlleris provided and wire-connected to the detector 19, the optical filters12, 16, and so forth, there can be performed automatically by thissystem controller the operations such that an electric potential to beapplied to the photoelectric multiplying tube 17 or intensity of thefluorescence excitation can be varied in accordance with intensity oflight to be introduced into the photoelectric multiplying tube 17 tomaintain the value of the photo-current flowing in the photoelectricmultiplying tube 17 in a range suitable for the photoelectricmultiplying tube 17, and, at the same time, the photo-current value withrespect to each fluorescence excitation intensity or each appliedvoltage is converted to a photo-current value with respect to a certaindefinite fluorescence intensity or a certain definite applied voltage.

Furthermore, in the above-described embodiment, explanations have beengiven as to a method for measuring, wherein the optical fiber 9 isdirectly introduced into the interior of the methane fermentation tank8, although it is also possible that the object for examination besampled from the methane fermentation tank and measured outside themethane fermentation tank. For example, the object for examination issampled from the methane fermentation tank through a sampling line, andthe sampled object is supplied to a sample adjuster by means of asampler part having a pumping function. In the sample adjuster, theobject is adjusted to a condition suitable for measurement, and then,supplied to the optical measurement part. The measurement is conductedin the same manner as in the embodiment of FIG. 2. Thus, such a systemcan be constructed without using the optical fibers as shown in FIG. 2.When the object for examination is subjected to the alkaline treatment,or to dilution, or the substitution of the liquid component of theobject for examination for other solution, such sampling of the objectfor examination would facilitate its treatment.

In addition, when Methanogens are immobilized to a immobilizing carrier,it is also possible to measure the object for examination at a positionof immobilized Methanogens by use of the optical fiber 9.

In the foregoing explanations of the invention, the measurement of thecell counts or the methane producing activity of Methanogens existing ina multitude of microorganism groups and foreign substances such asdigested sludge, etc. within the methane fermentation tank has beendescribed in the main, although the invention is not limited to suchobject for examination.

As described in the foregoing, the present invention makes it possibleto measure the cell counts or the methane producing activity ofMethanogens based on measurement of intensity of fluorescence in aparticular wavelength range to be radiated from the above-mentionedobject for examination by irradiation of excitation light of aparticular wavelength range onto the object for examination containingtherein Methanogens. In particular, even from the mixed system ofmicroorganism containing therein foreign substances, measurement of thecell counts or the methane producing activity of Methanogens can be doneeffectively.

INDUSTRIAL UTILITY

The present invention is applicable to measurement of the cell counts orthe methane producing activity of Methanogens existing in a multitude ofmicroorganism groups and foreign substances such as digested sludge,etc. in the methane fermentation tank, etc. for the waste water treatingsystem.

We claim:
 1. An apparatus for measuring cell counts or methane producingactivity of Methanogens contained within a vessel comprising:a lightsource means for generating light of a particular wavelength; a firstoptical fiber means operatively associated with said light source meansfor transmitting the light from said light source means into said vesselcausing the Methanogens to fluoresce; a second optical fiber meanshaving one end disposed inside said pressure vessel for collecting thelight flowered by the Methanogens contained within said vessel; aphoto-electric multiplying means operatively associated with the end ofsaid second optical fiber opposite that end of said second optical fibermeans which is disposed within said vessel; and a detector coupled tosaid photo-electric multiplying means for measuring the photo currentproduced by said photo-electric multiplying means.
 2. An apparatus as inclaim 1 wherein a filter is disposed between said light source means andsaid first optical fiber means for passing light having a wavelength of220 nm to 310 nm.
 3. An apparatus as in claim 1 wherein a filter isdisposed between said light source means and said first optical fibermeans for passing light having a wavelength of 220 nm to 255 nm.
 4. Anapparatus as in claim 1 wherein a filter is disposed between said lightsource means and said first optical fiber means for passing light havinga wavelength of 260 nm to 305 nm.
 5. An apparatus as in claim 1 whereina filter is disposed between said light source means and said firstoptical fiber means for passing light having a wavelength of 330 nm to370 nm.
 6. An apparatus as in claim 1 wherein a filter is disposedbetween said light source means and said first optical fiber means forpassing light having a wavelength of 380 nm to 440 nm.
 7. An apparatusas in claim 1 wherein a filter is disposed between said second opticalfiber means and said photo-elective multiplying means for passing lighthave a wavelength of 450 nm to 490 nm.
 8. An apparatus as in claim 1,further comprising:means for rendering alkaline the material within asample adjuster.
 9. An apparatus as in claim 1 further comprising:meansfor substituting the liquid component of the material contained within asample adjuster with a solution that does not fluoresce in the measuredwavelength range.
 10. An apparatus as in claim 1 for measuring cellcounts or methane producing activity of Methanogens contained within avessel further comprising:a sampling line means for sampling thematerial within said vessel; a sample adjuster means for adjusting saidmaterial to a condition suitable for measurement; an optical measurementpart means for measuring the light fluoresced by the Methanogens in thematerial; and a sampler part having a pumping function means forsampling the material from said vessel and supplying the material tosaid sample adjuster and optical measurement part.
 11. A method formeasuring cell counts or methane producing activity of Methanogens,characterized in that excited light having a wavelength range of from380 nm to 440 nm is irradiated onto a material containing thereinMethanogens, and then intensity of the light fluoresced by theMethanogens in the material, is measured, thereby obtaining the cellcounts or the methane producing activity of said Methanogens, whereinthe material is rendered alkaline and/or the liquid component of saidmaterial is substituted by a solution which does not fluoresce in themeasured wavelength range in the excited wavelength range.
 12. A methodfor measuring cell counts or methane producing activity of Methanogens,characterized in that excited light having a wavelength range of from380 nm to 440 nm is irradiated onto a material containing thereinMethanogens, and then intensity of the light fluoresced by theMethanogens in the material, is measured, thereby obtaining the cellcounts or the methane producing activity of said Methanogens, whereinthe material is rendered alkaline and/or the material is diluted with asolution which does not fluoresce in the measured wavelength range inthe excited wavelength range.